Identification and separation method of human hair follicle dermal stem cells

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to an identification and separation method of human hair follicle dermal stem cells. On the first aspect, the invention provides a marker of human hair follicle dermal stem cells, and the marker comprises one or more of COL11A1, RBP4, MMP11 and SOX2. A human hair follicle sample is directly obtained, a space transcriptome and a single cell RNA sequencing technology are combined, and a gene expression profile of a dermis part of the human hair follicle is comprehensively captured, so that the characteristic gene expression condition of the hHFDSC is identified. And meanwhile, positioning the structure position information of the hHFDSC by utilizing a space transcriptome, and analyzing the space distribution characteristics of the hHFDSC. By combining the two technologies, hHFDSC is definitely defined according to the identified feature gene and in combination with spatial positioning and differentiation capabilities of the feature gene, and efficient and accurate analysis of features and functions of hHFDSC is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for identifying and isolating human hair follicle dermal stem cells. Background Art

[0002] The hair follicle (HF) is a micro-organ in the human body with significant regenerative potential. Its structure is composed of epithelial cells and dermal cells. Adult hair follicles regenerate in a cyclical cycle of growth, regression and rest, continuously renewing the cells in the hair follicle, thereby achieving continuous regeneration and renewal of hair. The cyclical regeneration of hair follicles depends on a population of adult stem cells located in a specific ecological niche. They achieve self-renewal through proliferation and further differentiate into progeny cells to jointly maintain the complete structure of the hair follicle. In the dermis of the hair follicle, the cells are adjacent to the epithelial structure and can be subdivided into dermal papilla (DP) cells and dermal sheath (DS) cells. Studies have shown that DP cells play the role of a signaling center in the hair follicle microenvironment, regulating the proliferation and differentiation of epithelial cells. DS cells are wrapped around the outside of the hair follicle and play an important regulatory role in the hair follicle regeneration cycle.

[0003] Studies have revealed that there is a hair follicle dermal stem cell (HFDSC) at the base of mouse hair follicles. This stem cell significantly overexpresses Cdk1, Hic1, S100a4, and Itga8, is able to renew itself, and has the ability to differentiate into DP and DS cells, supporting the renewal of the dermal part of the hair follicle. HFDSC plays a key role in the regeneration of hair follicles. If the proliferation and differentiation functions of HFDSC are impaired, the renewal of the dermal part of the hair follicle will stagnate, and the regeneration activity of the hair follicle will not be completed. However, due to the species differences between humans and mice, the characteristic genes of mouse HFDSC cannot be located in the same hair follicle area in human hair follicles, nor can cells with similar functions be identified. At the same time, due to the lack of systematic research on human hair follicle dermal stem cells (hHFDSC), the existence of dermal stem cells has not yet been identified in human hair follicles.

[0004] In addition, the study also found that there are DP precursor cells, dermal sheath cup cells (DSCC), in the lower half of the dermal sheath of human hair follicles, close to the hair bulb of the hair follicle. The spatial position of DSCC is consistent with HFDSC, but the characteristic genes of DSCC are obviously different from HFDSC, and DSCC only supports the renewal and differentiation of its own cells into DP cells, and cannot differentiate into DS cells like HFDSC, making it difficult to complete the full renewal of the dermal part of the hair follicle. It can be seen that DSCC cannot play the role of hHFDSC. Therefore, it is necessary to further study and explore the characteristic genes that can accurately identify hHFDSC and develop a method for efficiently isolating hHFDSC. Summary of the invention

[0005] The present invention provides a method for identifying and isolating human hair follicle dermal stem cells. The characteristic genes relied on by the identification method can be used to effectively identify hHFDSCs, and the separation method can be used to efficiently isolate hHFDSCs.

[0006] In a first aspect of the present invention, a marker for identifying human hair follicle dermal stem cells is provided, wherein the marker comprises two or more of COL11A1, RBP4, MMP11 and SOX2.

[0007] In some embodiments of the present invention, the markers further include at least one of COL3A1, ASPN, LEF1, HAPLN1, POSTN, MME, TNMD, DPEP1, DKK3, TMEM119, CPXM2, SFRP1, LTBP2, SMOC2, COL8A2, FGFBP2, LAMC3, FAM180A, COL15A1, F2RL2, LRRC17 and TSPAN8.

[0008] A second aspect of the present invention provides a marker detection kit, which comprises a molecule that specifically recognizes the marker.

[0009] In some embodiments of the present invention, the molecule comprises at least one of an antibody, a primer, and a probe.

[0010] In some embodiments of the invention, the molecule is conjugated to a detectable label.

[0011] In some embodiments of the present invention, the detectable marker comprises at least one of a fluorescent marker, a chemiluminescent marker, a radioactive marker, an enzyme, biotin, and digoxigenin.

[0012] The third aspect of the present invention provides a method for identifying human hair follicle dermal stem cells, the method comprising detecting whether the aforementioned markers are expressed in the cells to be identified.

[0013] A fourth aspect of the present invention provides a method for screening human hair follicle dermal stem cells, the method comprising screening cells expressing the aforementioned markers in a cell population.

[0014] In some embodiments of the present invention, the screening is performed using the aforementioned detection kit.

[0015] In some embodiments of the invention, the cell population is derived from skin.

[0016] In some embodiments of the invention, the cell population is derived from hair follicles.

[0017] The fifth aspect of the present invention provides the use of the aforementioned detection kit in the preparation of products for identifying, screening and preparing human hair follicle dermal stem cells.

[0018] A sixth aspect of the present invention provides a method for isolating human hair follicle dermal stem cells, the method comprising the following steps: Separate the dermis of the hair bulb of the hair follicle; Digesting the dermis with type I collagenase to obtain a digestion product; The digestion product is subjected to a first centrifugation to obtain a supernatant containing the human hair follicle dermal stem cells, and the centrifugal force of the first centrifugation is 5 g to 30 g.

[0019] In some embodiments of the present invention, the method further comprises subjecting the supernatant to a second centrifugation to collect the precipitate, which is the human hair follicle dermal stem cells, and the centrifugal force of the second centrifugation is 200-400 g.

[0020] In some embodiments of the present invention, the supernatant further comprises cell filtration before the second centrifugation.

[0021] In some embodiments of the present invention, the working concentration of type I collagenase is 0.8-1.5 mg / mL.

[0022] In a seventh aspect, the present invention provides a method for producing human hair follicle dermal stem cells, the method comprising obtaining human hair follicle dermal stem cells according to the aforementioned method and culturing them.

[0023] In some embodiments of the present invention, the human hair follicle dermal stem cells obtained are genetically engineered to reduce immunogenicity.

[0024] The eighth aspect of the present invention provides a method for preparing cell secretions, which comprises obtaining human hair follicle dermal stem cells according to the aforementioned method and culturing them, and isolating the cell secretions.

[0025] In some embodiments of the present invention, the cell secretions include at least one of extracellular vesicles and cytokines.

[0026] The ninth aspect of the present invention provides a use of human hair follicle dermal stem cells obtained by the aforementioned method, or cell secretions obtained by the aforementioned preparation method in the preparation of products for reconstructing skin or hair follicles, promoting hair growth, and treating skin diseases.

[0027] The beneficial effects of the present invention are: In order to solve the problem of dermal stem cell identification caused by species differences, human hair follicle samples were directly obtained, and spatial transcriptome and single-cell RNA sequencing technology were combined to comprehensively capture the gene expression spectrum of the dermis of human hair follicles, thereby identifying the characteristic gene expression of hHFDSC. At the same time, spatial transcriptome was used to locate the structural position information of hHFDSC and analyze the spatial distribution characteristics of hHFDSC. By combining these two technologies, hHFDSC was clearly defined based on the identified characteristic genes, combined with its spatial positioning and differentiation ability, to achieve efficient and accurate analysis of the characteristics and functions of hHFDSC.

[0028] In addition, the present invention has also developed an efficient hHFDSC isolation method. The isolated cells have been confirmed to retain relatively complete biological functions through activity and functional evaluation. The entire isolation method has high specificity and high sensitivity, improves the purity and activity of cells, and optimizes the isolation operation process of human hair follicle dermal stem cells.

[0029] At the same time, the isolated hHFDSC or its extract can be further used for hair loss treatment in the form of lyophilized powder, exosomes, cell transplantation, culture medium, or combined with a delivery system, providing a new, safe and effective treatment method for hair loss patients, which has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a bubble chart of the expression levels of characteristic expression genes of hHFDSC in Example 1 of the present invention, wherein the average expression level represents the average expression level of a specific gene in hHFDSC / DP / DS cells, and the expression percentage represents the proportion of cells expressing a specific gene in hHFDSC / DP / DS cells in this type of cells.

[0031] Figure 2 It is the spatial position of hHFDSC in the spatial transcriptome data in Example 1 of the present invention.

[0032] Figure 3 This is a fluorescence staining image of hHFDSC-specific antibody in Example 1 of the present invention.

[0033] Figure 4These are the pseudo-time series results and RNA rate analysis results of human hair follicle dermal lineage cells in Example 1 of the present invention, wherein A is the Dynamo analysis result and B is the Monocle3 analysis result.

[0034] Figure 5 This is the result of obtaining and isolating hHFDSC in Example 2 of the present invention. A is the hair follicle unit obtained from the hospital, B is the hair bulb at the end of the cut hair follicle, C is the remaining dermis after removing the pigment and hair follicle keratin from the hair bulb, and D is the compact DP and discrete hHFDSC cell suspension after enzyme digestion.

[0035] Figure 6 These are the microscopic examination results of the primary and passaged hHFDSC cells in Examples 2 and 3 of the present invention. A is the cell morphology and activity of the primary hHFDSC cultured in the culture medium for three days, and B is the cell morphology and activity of the hHFDSC cultured once in the culture medium for three days.

[0036] Figure 7 It is the expression of the marker gene verified in Example 3 of the present invention in the isolated hHFDSC cells. Among them, A is the co-staining result of MMP11 and COL3A1 (20X), B is the co-staining result of MMP11 and RBP4 (20X), C is the co-staining result of COL11A1 and COL1A1 (20X), and D is the co-staining result of SOX2 and RBP4 (20X).

[0037] Figure 8 These are the experimental results of hHFDSC differentiation into DP cells in Example 3 of the present invention. A shows the cell morphology and activity (10X) of hHFDSC cultured once in culture medium for three days, B shows the cell morphology and activity (10X) of DP cells differentiated from hHFDSC, and C shows the co-staining result of RBP4 and AQP1 (20X).

[0038] Fig. 9 These are the experimental results of hHFDSC differentiation into DS cells in Example 3 of the present invention. A is the cell morphology and activity (10X) of hHFDSC cultured once in culture medium for three days, B is the cell morphology and activity (10X) of DS cells differentiated from hHFDSC, and C is the SMA staining result (20X). DETAILED DESCRIPTION

[0039] In the description of the present invention, the terms "first", "second", and "third" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first", "second", and "third" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0040] In a first aspect of the present invention, a marker for identifying human hair follicle dermal stem cells is provided, comprising multiple markers selected from COL11A1, RBP4, MMP11 and SOX2.

[0041] The term "hair follicle" refers to the sheath-like structure surrounding the hair root, which is divided into two layers: the inner layer is a stratified epithelium that is continuous with the epidermis, called the epithelial sheath (including the inner root sheath and the outer root sheath), and the outer layer is a connective tissue that is continuous with the dermis, called the connective tissue sheath. The end of the hair follicle swells to form a hair bulb. The bottom surface of the hair bulb is concave to form a pit, in which there is connective tissue, and blood vessels and nerve endings protrude in a papilla-like shape, called the hair papilla. Hair follicle dermal stem cells (HFDSCs) refer to a type of stem cells that support the renewal of the dermis of the hair follicle, can renew their own cells, and have the ability to differentiate into dermal papilla (DP) cells and dermal sheath (DS) cells. Usually, HFDSCs are located at the bottom of the hair bulb of the hair follicle, near the hair papilla. Accordingly, human hair follicle dermal stem cells refer to hair follicle dermal stem cells derived from humans, including hair follicle dermal stem cells directly isolated from human hair follicles, and also include cells after culture and passage (such as P1~P50 generations, such as P2, P3, P4, P5, P10, P20, P30, P40, P50). In addition, human hair follicle dermal stem cells also include cells differentiated from stem cells that meet their morphological characteristics, differentiation potential and express the same markers. The stem cells used to differentiate into human hair follicle dermal stem cells can be stem cells of non-embryonic origin, such as induced pluripotent stem cells (iPSCs). It is understandable that human hair follicle dermal stem cells can also be cells that have the same morphological characteristics, differentiation potential and expression of the same markers as the above-mentioned human hair follicle dermal stem cells after genetic engineering or other treatment methods (such as growth factor pretreatment, small molecule compound pretreatment, such as step-by-step regulation by co-treatment with bFGF and BMP inhibitor LDN-193189, see Nature 582, 399-404 (2020)). The purpose of the transformation or treatment can be, for example, to improve the proliferation, differentiation and hair follicle regeneration capabilities of the cells. In some embodiments, the human hair follicle dermal stem cells are adult stem cells, that is, hair follicle dermal stem cells derived from adult humans over 18 years old.

[0042] In addition, human hair follicle dermal stem cells can be one or more of autologous cells and allogeneic cells. Among them, autologous and allogeneic refer to the individual on whom the human hair follicle dermal stem cells act, that is, autologous cells are human hair follicle dermal stem cells derived from the individual itself, and allogeneic cells are human hair follicle dermal stem cells derived from other individuals other than the individual itself. When autologous cells are used, immune rejection reactions can be completely avoided during the action process. In some embodiments, when human hair follicle dermal stem cells are allogeneic cells, the cells can be modified in advance before the action to reduce their immunogenicity, such as using gene editing technology to modify allogeneic cells, or subjecting the individual to local or systemic immunosuppression.

[0043] The term "marker" refers to a biochemical index that can specifically mark the structure and function of a system, organ, tissue, cell, or subcell, as well as changes in structure and function or possible changes. Specifically, in this application, it refers to a marker that can specifically mark human hair follicle dermal stem cells, thereby distinguishing them from other cells. The marker can be one or more of DNA, RNA, and protein.

[0044] The marker includes multiple markers of COL11A1, RBP4, MMP11 and SOX2, for example, two, three or four of the markers, specifically, the marker includes COL11A1 and RBP4, the marker includes COL11A1 and MMP11, the marker includes COL11A1 and SOX2, the marker includes RBP4 and MMP11, the marker includes RBP4 and SOX2, the marker includes MMP11 and SOX2, the marker includes COL11A1, RBP4 and MMP11, the marker includes COL11A1, RBP4 and SOX2, the marker includes COL11A1, MMP11 and SOX2, the marker includes RBP4, MMP11 and SOX2, or the marker includes COL11A1, RBP4, MMP11 and SOX2. It is understood that the marker includes at least one of the above genes and the proteins encoded by the above genes. Thus, in some embodiments, the marker comprises genes COL11A1 and RBP4, the marker comprises genes COL11A1 and MMP11, the marker comprises genes COL11A1 and SOX2, the marker comprises genes RBP4 and MMP11, the marker comprises genes RBP4 and SOX2, the marker comprises genes MMP11 and SOX2, the marker comprises genes COL11A1, RBP4 and MMP11, the marker comprises genes COL11A1, RBP4 and SOX2, the marker comprises genes COL11A1, MMP11 and SOX2, the marker comprises genes RBP4, MMP11 and SOX2, or the marker comprises genes COL11A1, RBP4, MMP11 and SOX2. In other embodiments, the markers include COL11A1 protein and RBP4 protein, the markers include COL11A1 protein and MMP11 protein, the markers include COL11A1 protein and SOX2 protein, the markers include RBP4 protein and MMP11 protein, the markers include RBP4 protein and SOX2 protein, the markers include MMP11 protein and SOX2 protein, the markers include COL11A1 protein, RBP4 protein and MMP11 protein, the markers include COL11A1 protein, RBP4 protein and SOX2 protein, the markers include COL11A1 protein, MMP11 protein and SOX2 protein, the markers include RBP4 protein, MMP11 protein and SOX2 protein, or the markers include COL11A1 protein, RBP4 protein, MMP11 protein and SOX2 protein.

[0045] In some embodiments, the markers further include at least one of set A, which is a set consisting of COL3A1, ASPN, LEF1, HAPLN1, POSTN, MME, TNMD, DPEP1, DKK3, TMEM119, CPXM2, SFRP1, LTBP2, SMOC2, COL8A2, FGFBP2, LAMC3, FAM180A, COL15A1, F2RL2, LRRC17, and TSPAN8. In some embodiments, the markers further include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or all 22 of set A.

[0046] In some embodiments, the markers further include COL3A1. In some embodiments, the markers further include ASPN. In some embodiments, the markers further include LEF1. In some embodiments, the markers further include HAPLN1. In some embodiments, the markers further include POSTN. In some embodiments, the markers further include MME. In some embodiments, the markers further include TNMD. In some embodiments, the markers further include DPEP1. In some embodiments, the markers further include DKK3. In some embodiments, the markers further include TMEM119. In some embodiments, the markers further include CPXM2. In some embodiments, the markers further include SFRP1. In some embodiments, the markers further include LTBP2. In some embodiments, the markers further include SMOC2. In some embodiments, the markers further include COL8A2. In some embodiments, the markers further include FGFBP2. In some embodiments, the markers further include LAMC3. In some embodiments, the markers further include FAM180A. In some embodiments, the markers further include COL15A1. In some embodiments, the markers further include F2RL2. In some embodiments, the markers further include LRRC17. In some embodiments, the marker further comprises TSPAN 8. It is understood that the markers in the above set A may also be at least one of genes or proteins.

[0047] The second aspect of the present invention provides a marker detection kit, which includes a molecule that specifically recognizes the marker.

[0048] In some embodiments, the molecule is detected by immunohistochemistry, enzyme-linked immunosorbent assay, radioimmunoassay, immunoprecipitation, immunochemiluminescence, colloidal gold immunoassay, fluorescent immunochromatography, complement fixation analysis, flow cytometry fluorescence resolution, single molecule detection technology, Western blotting, Northern blotting, amplification (such as polymerase chain reaction PCR, and isothermal amplification such as loop-mediated isothermal amplification LAMP, recombinase polymerase amplification RPA, rolling circle amplification RCA, cross primer amplification CPA, strand displacement amplification The marker is detected by one or more of the following techniques: SDA, helicase-dependent amplification HDA), biochip method, nucleic acid sequencing method, amino acid sequencing method, high performance liquid chromatography, capillary gel electrophoresis, near infrared spectroscopy, mass spectrometry (such as electron impact mass spectrometry, chemical ionization mass spectrometry, photoionization mass spectrometry, threshold ionization mass spectrometry, double focusing mass spectrometry, quadrupole mass spectrometry, time-of-flight mass spectrometry, ion trap mass spectrometry, Fourier transform mass spectrometry, Fourier transform ion cyclotron resonance mass spectrometry), surface plasmon resonance technology, immuno-PCR technology, biotin-avidin technology, etc. It is understandable that the type of molecules specifically recognized may also be different depending on the specific type of the marker. For example, in some embodiments, the molecules of each marker in the specific recognition marker can be any one of an antibody that can specifically bind to the marker, a ligand protein or polypeptide that specifically binds to the marker, a non-protein compound that specifically recognizes the marker, a probe that specifically binds to the marker, and a nucleic acid aptamer that specifically recognizes the marker.

[0049] In some embodiments, the antibody also includes an antigen-binding fragment. Specifically, the antibody can be at least one of a polyclonal antibody, a monoclonal antibody, a single-chain antibody, a functional antibody fragment, an antibody Fab region, a nanobody, a chimeric antibody, and a multispecific antibody.

[0050] In some embodiments, the molecule is coupled to a detectable marker. In some embodiments, the detectable marker includes at least one of a fluorescent marker, a chemiluminescent marker, a radioactive marker, an enzyme, biotin, and digoxin. In some embodiments, the fluorescent marker includes a fluorescent dye, such as cyanines (e.g., Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5, Alexa Fluor 555, etc.), rhodamines (e.g., rhodamine 101, rhodamine 6G, tetramethylrhodamine, rhodamine B, X-rhodamine, Texas Red, Alexa Fluor 488, Alexa Fluor 594, etc.), fluoresceins (e.g., fluorescein, fluorescein isothiocyanate, 5 (6)-carboxyhexachlorofluorescein, 5 (6)-carboxynaphthylfluorescein, Oregon Green, Pennsylvania Green, etc.), coumarins (e.g., 7-AMCA, Alexa Fluor 350, Coumarin 343, etc.), fluorescent proteins (e.g., green fluorescent protein, phycoerythrin, phycocyanin, allophycocyanin), quantum dots (IV group quantum dots, such as C, Si, Ge; III-V group quantum dots, such as InAs, InP, GaSb, GaN, GaAs, InGaAs, AlGaAs, InAlAs, InGaN; II-VI group quantum dots, such as BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe; IV-IV group quantum dots, such as SiC, SiGe; IV-VI group quantum dots, such as PbS, PbSe), nanocrystals, etc. In some embodiments, chemiluminescent labels include acridinium esters, ruthenium pyridine, luminol and its derivatives (such as isoluminol), etc. In some embodiments, radioactive labels include tritium, carbon 14, phosphorus 32, sulfur 35, iodine 125, iodine 131, etc. In some embodiments, the enzyme includes horseradish peroxidase (HRP), alkaline phosphatase (ALP), and the like.

[0051] In some embodiments, the detection kit further comprises reagents for assisting the detection of gene expression, such as reaction reagents that can visualize the amplicon corresponding to the primer, RNA extraction reagents, reverse transcription reagents, cDNA amplification reagents, standard products used to prepare standard curves, and at least one of positive controls. In some embodiments, the detection kit further comprises reagents for assisting the detection of protein expression, such as color developers, blocking solutions, antibody diluents, washing buffers, color stop solutions, standard products used to prepare standard curves, and at least one of positive controls.

[0052] The third aspect of the present invention provides a method for identifying human hair follicle dermal stem cells, the method comprising detecting whether the aforementioned markers are expressed in the cells to be identified.

[0053] In some embodiments of the present invention, the detection is performed using the aforementioned detection kit.

[0054] In some embodiments, the cells to be identified are derived from skin. In some embodiments, the cells to be identified are derived from hair follicles.

[0055] In some embodiments, the total number of cells in the cells to be identified can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 10 14 10 15 10 16 10 17 10 18 10 19 10 20 It is understandable that, depending on the different detection methods, one or more cells in the cells to be identified can be identified simultaneously or successively.

[0056] A fourth aspect of the present invention provides a method for screening human hair follicle dermal stem cells, the method comprising screening cells expressing the aforementioned markers in a cell population.

[0057] In some embodiments, the screening is performed by the aforementioned detection kit.

[0058] In some embodiments, the cell population is derived from skin. In some embodiments, the cell population is derived from hair follicles.

[0059] In some embodiments, the total number of cells in the cell population can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 1210 13 10 14 10 15 10 16 10 17 10 18 10 19 10 20 More than one.

[0060] In some embodiments, screening or identifying is performed by flow cytometry.

[0061] In a fifth aspect of the present invention, the use of the aforementioned detection kit in preparing a product for identifying, screening, and preparing human hair follicle dermal stem cells is provided. In some embodiments, the product includes any one of a chip, a device, and a system, for example, a chip, a device, or a system for identifying, screening, and preparing human hair follicle dermal stem cells.

[0062] A sixth aspect of the present invention provides a method for isolating human hair follicle dermal stem cells, the method comprising the following steps: Separate the dermis of the hair bulb of the hair follicle; The dermis was digested with type I collagenase to obtain the digestion product; The digestion product is subjected to a first centrifugation to obtain a supernatant containing human hair follicle dermal stem cells.

[0063] In some embodiments, the dermis is digested in an enzyme solution of 0.1-10 mg / mL type I collagenase, and the working concentration of the enzyme solution can be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL. In some embodiments, the dermis is digested in an enzyme solution of type I collagenase at a working concentration of 0.8-1.5 mg / mL. In some embodiments, the digestion temperature is 4-40°C, for example, 4°C, 6°C, 8°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C. In some embodiments, the digestion temperature is 35-40°C. In some embodiments, the digestion time is 10 min-10 h, for example, 10 min, 20 min, 30 min, 40 min, 45 min, 50 min, 55 min, 60 min, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h. In some embodiments, the digestion time is 50 min-60 min. Wherein, type I collagenase is an enzyme mixture, the main components of which include clostripain, neutral protease and two collagenases, thereby degrading collagen and other intracellular substances. Human hair follicle dermal stem cells are dermal fibroblasts of hair follicles, and compared with other collagenases or other types of enzymes, type I collagenase can effectively digest collagen in the extracellular matrix, especially type I collagen, which is the most important type of collagen in the dermis of hair follicles, so that better digestion and separation effects can be achieved during the separation process. One or more enzymes in type I collagenase can be wild-type enzymes, or products modified by genetic engineering on the basis of wild-type enzymes, such as mutant enzymes obtained by random mutation, site-directed mutation, DNA shuffling, etc. for one or more purposes such as improving enzyme activity and thermal stability, and have similar or higher ability to effectively release cells from tissues. In some embodiments, after the hair bulb is digested with type I collagenase, the digestion product is further subjected to a first centrifugation step.

[0064] In some embodiments, the centrifugal force of the first centrifugation is above 5 g, specifically 5 to 30 g, such as 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g, 13 g, 14 g, 15 g, 16 g, 18 g, 20 g, 25 g, 30 g, etc. When the centrifugal force of the first centrifugation is too low, the separation effect is not ideal, and it is difficult to clearly distinguish the supernatant containing HFDSC and the precipitate containing DP cell mammospheres; when the centrifugal force is too large, it is helpful for separation, but the precipitate of DP cell mammospheres may be damaged. In some embodiments, the time of the first centrifugation is 1 to 5 min, such as 1 min, 2 min, 3 min, 4 min, 5 min.

[0065] In some embodiments, the supernatant containing human hair follicle dermal stem cells is filtered. In some embodiments, after the first centrifugation, a filter with a mesh diameter of 20 to 60 μm is used to filter out impurities and larger cell clumps, for example, the mesh diameter can be 20 μm, 30 μm, 40 μm, 50 μm, or 60 μm. In some embodiments, the filter is a cell sieve, and the specification of the cell sieve is 40 μm.

[0066] In some embodiments, the supernatant is further centrifuged for a second time, and the collected precipitate is human hair follicle dermal stem cells. In some embodiments, the centrifugal force of the second centrifugation is 200 g or more, specifically 200-400 g, such as 200 g, 220 g, 240 g, 250 g, 260 g, 280 g, 300 g, 320 g, 340 g, 350 g, 360 g, 380 g, 400 g. In some embodiments, the second centrifugation time is 2-10 min, such as 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min.

[0067] In some embodiments, the bulb of the hair follicle is the portion of 0.1 to 2 mm at the end of the hair follicle, for example, it can be 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm. In some embodiments, the pigment and keratin in the bulb are removed to obtain the dermis of the bulb. In some embodiments, the pigment and keratin in the bulb are removed by mechanical extrusion.

[0068] In a seventh aspect, the present invention provides a method for producing human hair follicle dermal stem cells, the method comprising obtaining human hair follicle dermal stem cells according to the aforementioned method and culturing them.

[0069] The term "culture" refers to the process in which cells grow and proliferate under certain conditions to produce a large number of cells. In some embodiments, the culture can be 2D culture or 3D culture. In some embodiments, the 3D culture method includes 3D culture with a scaffold or 3D culture without a scaffold. In some embodiments, the 3D culture method without a scaffold includes at least one of a low adhesion plane method, a hanging drop method, a stirring method, a 3D culture dish, etc. In some embodiments, the scaffold material for 3D culture can be one or more of bioceramics, bioglass, metal, natural polymers (such as alginate, silk, hyaluronic acid, chitosan, silk fibroin, fibrin, collagen, pluronic acid, etc.) and synthetic polymers (such as polylactic acid, polyglycolic acid, polycaprolactone, polyacrylonitrile, polyethylene glycol, polyimide, polyvinyl alcohol, etc.). In some embodiments, the culture time is 10 to 60 days, for example, it can be 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days. In some embodiments, the culture is performed in at least one of a culture dish, a culture vessel, a cell factory, a microfluidic chip, and the like.

[0070] In some embodiments, the obtained human hair follicle dermal stem cells are genetically engineered to reduce immunogenicity.

[0071] In an eighth aspect of the present invention, a method for preparing cell secretions is provided, the preparation method comprising obtaining human hair follicle dermal stem cells according to the aforementioned method and culturing them, and isolating cell secretions. In some embodiments, the cell secretions include at least one of extracellular vesicles (such as exosomes, microvesicles, etc.) and cytokines. In some embodiments, the method for isolating extracellular vesicles includes one or more of centrifugation (such as ultracentrifugation), ultrafiltration, size exclusion chromatography, precipitation, immunoaffinity, microfluidics, etc. In some embodiments, isolating cytokines includes taking the supernatant of the culture product.

[0072] In the ninth aspect of the present invention, there is provided a human hair follicle dermal stem cell obtained by the aforementioned method, or a cell secretion obtained by the aforementioned preparation method, for use in preparing products for reconstructing skin or hair follicles, promoting hair growth, and treating skin diseases. Due to the differentiation potential of human hair follicle dermal stem cells, DP cells play the role of a signal center in the hair follicle microenvironment, regulating the proliferation and differentiation of epithelial cells, and DS cells are wrapped around the outside of the hair follicles and have an important regulatory effect on the hair follicle regeneration cycle. Therefore, human hair follicle dermal stem cells may have a corresponding effect on reconstructing skin or hair follicles, promoting hair growth, and treating skin diseases.

[0073] In some embodiments, the product for reconstructing skin or hair follicles, promoting hair growth, and treating skin diseases also includes a drug for promoting hair growth, such as minoxidil, finasteride, etc. In some embodiments, the multiple components in the product for reconstructing skin or hair follicles, promoting hair growth, and treating skin diseases can be applied simultaneously or sequentially in one or more different ways. In some embodiments, the method of application can be one or more of external use or internal administration.

[0074] In some embodiments, skin diseases include vitiligo, scars, hair loss (such as androgenetic alopecia AGA and alopecia areata AA), etc.

[0075] In some embodiments, the product, when used, comprises applying human hair follicle dermal stem cells, or cell secretions, to a person's head.

[0076] The present invention is further described in detail below through specific examples.

[0077] It should be understood that these embodiments are only used to illustrate the present invention but are not used to limit the scope of the present invention.

[0078] Some preferred embodiments of the present invention are given below. The experimental methods without specific conditions are usually carried out under conventional conditions or the conditions recommended by the manufacturer. The human scalp tissues in the examples are from 22 healthy male volunteers (3-77 years old) who underwent scalp tumor resection surgery and observed under a Leica fluorescence microscope. Some conventional materials are shown in Table 1. The other materials, reagents, etc. used are also reagents and materials obtained from commercial sources unless otherwise specified.

[0079] Table 1. Some experimental materials and sources

[0080] Example 1: Identification of human hair follicle dermal stem cells This embodiment provides a method for identifying human hair follicle dermal stem cells, and the process is as follows: (1) Tissue sample acquisition and pretreatment: Obtain scalp tissue from the back of the head of a healthy person without hair loss, wash it with PBS, and remove excess subcutaneous fat. Cut the washed scalp tissue into small pieces with sterile ophthalmic scissors to provide materials for subsequent spatial transcriptome and single-cell sequencing.

[0081] (2) Spatial transcriptome sequencing experiment: Tissue samples were cut into small pieces and fixed with OCT according to the direction of hair growth and stored at -80 °C. After the embedded tissue blocks were balanced to -20 °C, they were cut to a thickness of 12 μm using a cryo-microtome, and the tissue sections were attached to the Stereo-seq chip and fixed with pre-cooled methanol (-20 °C) for 30 minutes. Subsequently, they were stained with ssDNA fluorescent dye for 5 minutes at room temperature in the dark, washed with 0.1 v / v% SSC buffer and air-dried, glycerol was applied to the tissue sections and placed with a cover glass. The staining was recorded using a fluorescence microscope, and the image quality was evaluated using ImageQC software. After imaging, the cover glass was removed from the chip, washed with 5 v / v% SSC buffer, and permeabilized with 0.1 v / v% pepsin at room temperature for 8 minutes. After permeabilization, in situ reverse transcription and cDNA amplification were completed on the chip at 42 °C, and finally sequenced using the DNBSEQ platform to obtain spatial transcriptome data. The read length of read 1 is 35 bp, and the read length of read 2 is 100 bp.

[0082] (3) scRNA-seq sequencing experiment: The tissues cut into small pieces were placed in 5 mL of preheated type I enzyme solution (4.0 mg / mL dispase II) and incubated at 37 °C for 50 minutes. The tissue suspension was transferred to the preheated type II enzyme solution (1.6 mg / mL type I and 2.0 mg / mL type IV collagenase) and incubated at 37 °C for 30 minutes. Then, 20 mL of DMEM was added to stop the enzymatic reaction, and the cell suspension was collected through a 70 μm cell strainer. The remaining undigested tissue fragments were digested with 8 mL of 0.25% trypsin at 37 °C for 15 minutes. The second digestion was stopped by adding 10 mL of DMEM containing 20 v / v% fetal bovine serum (FBS). The cell suspension was then filtered through a 70 μm cell strainer and the filtrate was mixed with the previously collected filtrate. All subsequent operations were performed on ice or at 4 °C. The mixed cell suspension was centrifuged and the cell pellet was resuspended in PBS containing 2 v / v% FBS. The suspension was then passed through a 30 μm cell strainer to remove any remaining debris. Finally, the filtered suspension was centrifuged and the cell pellet was resuspended in PBS containing 0.04 v / v% bovine serum albumin (BSA) to generate the final single-cell suspension. The MGI DNBelab C series high-throughput single-cell RNA library preparation process was used to generate single-cell transcriptome data after cell barcode labeling, reverse transcription, cDNA amplification, fragmentation, and library circularization, and sequencing was performed using the DNBSEQ platform.

[0083] (4) scRNA-seq data quality control: 1. Raw data processing: After quality filtering of the paired-end reads of the raw sequencing, the sequencing data were aligned to the human genome (hg38) using STAR (v2.7.1a). In order to screen single-cell droplets with valid cell barcodes, the EmptyDrops algorithm was used to analyze the original droplet-gene matrix by calculating the cosine similarity of oligonucleotide types and barcodes counts between droplets, and PISA (v0.2) was used to generate the cell-gene UMI count matrix.

[0084] 2. Removal of environmental free RNA: SoupX (v1.6.2) was used to remove environmental RNA contamination from single-cell RNA-seq data. First, a SoupChannel object was created using the original and filtered matrices. Clustering was then performed in the SoupChannel object, and the level of environmental RNA contamination was estimated using the autoEstCont function. Finally, the counts were adjusted to remove environmental RNA contamination using the adjustCounts function, and the results were rounded to integers.

[0085] 3. Doublet detection: scDblFinder (v1.12.0) was used to detect doublets in scRNA-seq data. First, a SingleCellExperiment object was created for the raw count data of each library. Subsequently, the scDblFinder function was run on each library with the following parameters: clusters=TRUE, aggregateFeatures=TRUE, nfeatures=25, processing="normFeatures". The doublet detection results were added to the Seurat object for further analysis.

[0086] 4. Data quality control: To ensure data quality, each sample was first filtered to remove cells with UMI counts below 100 (UMI < 100). Then, all samples were merged into a Seurat object, and the median and median absolute deviation (MAD) of all cells were calculated. Values ​​that deviated from the median by more than four times the MAD were marked as outliers. Finally, cells marked as outliers and double cells were removed from all sample libraries.

[0087] (5) Identification of genes specifically expressed in hHFDSCs: 1. After integrating batch effects, the Seurat software package was used to perform data dimension reduction and clustering, and the highly expressed characteristic genes of the cell population were captured based on the FindMarkers function. Genes that met p<0.05 and log2(Fold changes)>0.5 were screened.

[0088] 2. Through gene signatures, we screened out dermal cell populations (cell populations with high expression of THY1, COL1A1, TPM1, and PDGFRA), and further identified different subtypes in the dermal cell populations. Finally, we identified (annotated) DP cells, DS cells, and hHFDSCs. The results are as follows: Figure 1 The characteristic genes of hHFDSC include multiple genes of COL11A1, RBP4, MMP11, and SOX2, and also include one or more genes of highly expressed LEF1, HAPLN1, COL3A1, ASPN, POSTN, MME, TNMD, DPEP1, DKK3, TMEM119, CPXM2, SFRP1, LTBP2, SMOC2, COL8A2, FGFBP2, LAMC3, FAM180A, COL15A1, F2RL2, LRRC17, and TSPAN8.

[0089] (6) Spatial localization analysis of hHFDSC: 1. Process the raw data of spatial transcriptome sequencing, use STOmics Analysis Workflow (SAW v6.0) to align and denoise the sequencing data, and generate a UMI matrix with spatial coordinates.

[0090] 2. Use ssDNA staining images and RNA grayscale images for manual registration to accurately locate cell positions. Use Gaussian weighted local threshold and Euclidean distance transform algorithm to segment cells and generate cell labels. Summarize the UMIs in DNB to obtain the gene expression matrix at the cellular level.

[0091] 3. Combined with the cell type annotation data of scRNA-seq in (5), locate the spatial position of hHFDSC. The technical noise introduced in the spatial transcriptome data acquisition process may affect the detection of tissue transcript expression levels, thereby affecting the identification of cell types. In addition, compared with scNRA-seq, the transcript signals of the spatial transcriptome are relatively sparse, which makes the accurate identification of cell types somewhat complicated. Combined with the robust cell type decomposition (RCTD) method, the cell type annotation data of scRNA-seq in (5) was used to create a reference dataset, and the cells obtained by spatial transcriptome segmentation were labeled with cell types using supervised learning methods. The spatial expression matrix and coordinates were combined to determine the cell types of the spatial transcriptome. The results are shown in the figure. Figure 2 As shown, hHFDSCs are localized at the base of the hair bulb of the hair follicle, in the fusiform region adjacent to the DP cells.

[0092] (6) Immunofluorescence verification of hHFDSC characteristic genes: Human scalp tissue samples were fixed with 4 v / v% paraformaldehyde, embedded in paraffin, and sliced ​​with a thickness of 7 μm. The paraffin sections were dewaxed and antigen-recovered, and blocked with 5 v / v% BSA for 1 hour at room temperature. Antibodies to hHFDSC marker genes (MMP11+RBP4, SOX2+RBP4, COL11A1+RBP4) were added to the sections and incubated overnight at 4°C. After washing three times with PBS, fluorescent secondary antibodies (see Table 1 for details) were added and incubated at room temperature for 1 hour. The cell nuclei were counterstained with 10 mg / mL Hoechst 33342 at room temperature for 15 minutes, and images were collected using an OLYMPUS FV4000 confocal microscope to analyze the characteristic gene-positive cells. The results are shown in Figure 3 As shown, the locations of the positive cells stained by the four antibodies were consistent with the hHFDSC positions locked in the spatial transcriptome sequencing, confirming the validity of the spatial transcriptome sequencing data and the accuracy of the hHFDSC spatial position.

[0093] (7) Analysis of differentiation potential of hHFDSCs: 1. Based on Stereo-seq data, Dynamo was used to analyze the spatial RNA dynamic rate of hHFDSC. Dynamo calculated the ratio of uncut and cut RNA, established a gradient vector field for the transcriptome state of the cell as a continuous variable, estimated the dynamic change direction and rate of gene expression, and thus predicted the trend of gene expression changes in the future state of the cell and constructed a spatial lineage pseudo-time series trajectory. The results are as follows Figure 4 As shown in A, according to the Dynamo analysis results, hHFDSC is located at the bottom of the hair follicle bulb, and the direction of change in its gene expression points to DP cells (red) and DS cells (light gray) as shown by the arrows. The hHFDSC pseudo-time value is low and has the potential to differentiate into DP and DS cells.

[0094] 2. Use monocle3 to learn the pseudo-time series trajectory of scRNA-seq data. The results are as follows Figure 4 As shown in Figure B, the hHFDSC (green) cell population has a low pseudo-timing value and is at the starting point of the cell differentiation trajectory. As the pseudo-timing value of the cell population increases, hHFDSC can differentiate into DP and DS cells. The above results further verify that hHFDSC has the potential to differentiate into DP and DS cells, confirming its stem cell characteristics.

[0095] Example 2: Isolation of human hair follicle dermal stem cells This embodiment provides a method for isolating human hair follicle dermal stem cells, the steps are as follows: (1) Hair follicle acquisition Hair follicles from the occipital region were removed from the heads of patients undergoing craniotomy or hair transplantation in the hospital. Figure 5A, immediately placed on ice for temporary storage at low temperature after ex vivo, to maintain cell activity. The hair follicles were then quickly transported (using cold chain transportation equipment to control the low temperature environment during transportation) to the hospital laboratory for further processing. The hair follicles were washed with 1× PBS buffer, and the reagents were dried with dust-free paper after washing to prevent residual reagents from affecting the subsequent cell separation process. Pay attention to aseptic operation in this step to avoid contaminating the hair follicles.

[0096] (2) Isolation of hHFDSC 1. Hair follicle treatment: reference Figure 5 B~D, first cut off the hair follicle bulb 1 mm away from the end of the hair follicle to create an entrance for subsequent cell separation. Then use mechanical force to squeeze out the pigment and hair follicle keratin components in the hair follicle bulb to reduce the interference of non-target cells and impurities. The remaining follicle dermis after squeezing was digested with 1 mg / mL type I collagenase at 37°C for 55 minutes to achieve full dissociation of the tissue while maintaining the integrity of the cells. During the digestion process, the mixture needs to be gently blown at regular intervals. This operation helps the collagenase to fully contact the dermis of the hair follicle, improves the digestion efficiency, ensures full dissociation of the tissue, and lays the foundation for the subsequent hHFDSC cell separation and purification.

[0097] 2. Cell separation: The cell suspension obtained after digestion was centrifuged at low speed (10 g, 2 minutes) to separate the supernatant containing hHFDSC and the precipitate containing DP cell mammospheres. This low-speed centrifugation parameter can effectively separate the target cells while reducing mechanical damage to the cells. The separated supernatant was collected and filtered through a 40 μm cell sieve to remove impurities and larger cell aggregates in the cell suspension and improve the purity of the final cell sample. The filtered supernatant was then centrifuged at a centrifugal force of 300 g for 5 minutes to obtain purified hHFDSC (see Figure 5 The optimization of the centrifugation step ensures the high purity and activity of hHFDSC, providing high-quality cell samples for subsequent cell culture.

[0098] During the entire separation process, the operation needs to be gentle to avoid damage to hHFDSC. Through the above optimization method, this embodiment can provide an efficient and gentle cell separation method, which significantly improves the separation efficiency and cell viability of hHFDSC.

[0099] Example 3: Identification of isolated cells (1) Morphology, proliferation ability and activity identification The hHFDSC isolated in Example 2 was cultured in high-glucose DMEM medium (supplemented with 20 v / v% FBS, 1 v / v% penicillin / streptomycin, 1 v / v% non-essential amino acids (NEAA) and 1 v / v% GlutaMAX) at 37 °C and 5% CO2. The culture medium was replaced every 3 days to ensure that the cells obtain sufficient nutrients and growth factors to promote cell proliferation and maintain their activity. When replacing the culture medium, attention should be paid to the sterility of the operation to avoid contamination. At the same time, the above culture conditions simulate the physiological environment in the body, which is conducive to the normal growth and function of the cells.

[0100] When the cell density reaches 80%, the medium is discarded, the cells are washed with PBS, and 0.05 v / v% trypsin is added for digestion at 37°C for 3 minutes. The digestion is terminated by adding high-glucose DMEM medium containing 10 v / v% FBS. The cell suspension is transferred into a 15 mL centrifuge tube and centrifuged at 300 g for 3 minutes. When the cell pellet is visible, the supernatant is discarded, and the cell pellet is suspended with high-glucose DMEM medium (the added components are the same as above), counted, and inoculated into a new culture bottle at a density of 1×106 cells / mL and continued to be cultured under the same culture conditions. This method is repeated for passage.

[0101] The results are as follows Figure 6 As shown, the isolated primary hHFDSCs are basically consistent with the hHFDSCs after passage in morphology, proliferation ability and activity, and the experiment has strong repeatability. Therefore, the hHFDSCs isolated in the embodiment can be expanded normally and the cell quality is good.

[0102] (2) Identification of characteristic genes Example 2 The hHFDSCs isolated were blocked with 5 v / v% BSA in PBS at room temperature for 1 hour, the primary antibody of the marker gene was added, incubated at 4°C overnight, washed to remove the unbound antibody, the fluorescent secondary antibody was added, incubated at 4°C for 1 hour, washed to remove the unbound secondary antibody, and DAPI was added to counterstain the cell nucleus for 15 minutes. The image was collected using an OLYMPUS FV4000 confocal microscope and the characteristic gene-positive cells were analyzed. The results are shown in Figure 7 As shown, co-staining with MMP11+COL3A1, MMP11+RBP4, COL11A1+COL1A1, and SOX2+RBP4 respectively detected co-localized expression in hHFDSCs. Therefore, the cells isolated in Example 2 are hHFDSCs and have the expected molecular characteristics.

[0103] (3) Identification of differentiation potential 1. DP cell differentiation potential After the isolated hHFDSCs were cultured to P3, they were replaced with DMEM / F12 medium (supplemented with 10 v / v% FBS) and cultured for another generation. Immunofluorescence staining of AQP1 and RBP4 was performed in the same manner as in (2). Figure 8 As shown, under the microscope, it was found that the cell morphology became more slender, the expression of AQP1 protein, a marker of DP cells, was upregulated, and the expression of RBP4 was downregulated, showing in vitro differentiation characteristics. Therefore, hHFDSCs were successfully differentiated into DP cells.

[0104] 2. DS cell differentiation potential When hHFDSCs were cultured in high-glucose DMEM medium (with the same ingredients as above) to P4 and continued to be cultured for one more generation, immunofluorescence staining of SMA was performed in the same manner as in (2). Fig. 9 As shown in the figure, the cell morphology gradually changed from slender to flat, the number of antennae increased, the stem cell characteristics began to decline, and SMA protein began to be expressed. Therefore, hHFDSC cells have a tendency to differentiate into DS.

[0105] Example 4 This example provides a method for isolating human hair follicle dermal stem cells, which is different from Example 2 in that the centrifugal force used in the low-speed centrifugation is 20 g. The results show that although a larger centrifugal force is helpful for separation, it will damage the precipitation of DP cell mammospheres, which is not conducive to maintaining the integrity and cell viability of DP cell mammospheres.

[0106] Comparative Example 1 This comparative example provides a method for isolating human hair follicle dermal stem cells, which is different from Example 2 in that the centrifugal force used in the low-speed centrifugation is 4 g. The results show that it is difficult to clearly distinguish the supernatant of hHFDSC and the precipitate containing DP cell microspheres in this step, and the separation effect is not ideal.

[0107] Comparative Example 2 This comparative example provides a method for isolating human hair follicle dermal stem cells, which is different from Example 2 in that: in the separation process, type II collagenase, type III collagenase, type IV collagenase, pancreatin or neutral protease of the same enzyme concentration are used to replace type I collagenase. Type II collagenase usually has a strong digestion effect on the extracellular matrix, while type III and type IV collagenases may be more specific to collagen binding on the cell surface. Pancreatin mainly acts on proteins on the cell membrane, and neutral protease has a wider substrate specificity. The results show that the separation effect of hHFDSC is affected when these alternative enzymes are used for digestion. Compared with type I collagenase, these enzymes may cause excessive digestion of the extracellular matrix and cell membrane, thereby damaging the integrity of hHFDSC and affecting its vitality and subsequent function.

[0108] Comparative Example 3 This comparative example provides a method for isolating human hair follicle dermal stem cells, which is different from Example 2 in that the concentration of type I collagenase is adjusted to 0.5 mg / mL (low concentration) and 2 mg / mL (high concentration). The results show that the digestion ability of low-concentration collagenase is insufficient, resulting in incomplete separation of hHFDSC from surrounding tissues, affecting the purity of the cells; and although high-concentration collagenase enhances the digestion ability, it may destroy the structure of hHFDSC due to excessive digestion and reduce cell viability.

[0109] Comparative Example 4 This comparative example provides a method for isolating human hair follicle dermal stem cells, which differs from Example 2 in that the temperature of the digestion process is adjusted to 32 degrees Celsius (low temperature) and 42 degrees Celsius (high temperature). The results show that under low temperature conditions, the activity of collagenase is reduced, resulting in a slow digestion speed and poor separation effect; while under high temperature conditions, although the digestion speed is accelerated, it may cause thermal damage to hHFDSC, affecting the activity and function of the cells.

[0110] Comparative Example 5 This comparative example provides a method for isolating human hair follicle dermal stem cells, which differs from Example 2 in that the digestion time is adjusted to 45 minutes (short time) and 65 minutes (long time). The results show that short digestion time is not enough to fully isolate hHFDSC, resulting in low purity; while long digestion time may lead to over-digestion of cells, decreased cell viability, or even cell death.

[0111] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Identify markers of human hair follicle dermal stem cells, characterized in that: The markers include multiple ones of COL11A1, RBP4, MMP11 and SOX2.

2. The marker according to claim 1, characterized in that: The markers also include at least one of COL3A1, ASPN, LEF1, HAPLN1, POSTN, MME, TNMD, DPEP1, DKK3, TMEM119, CPXM2, SFRP1, LTBP2, SMOC2, COL8A2, FGFBP2, LAMC3, FAM180A, COL15A1, F2RL2, LRRC17 and TSPAN8.

3. The marker detection kit according to claim 1 or 2, characterized in that: The detection kit includes a molecule that specifically recognizes the marker.

4. The detection kit according to claim 3, characterized in that: The molecule includes at least one of an antibody, a primer, and a probe.

5. The detection kit according to claim 3, characterized in that: The molecule is coupled to a detectable label.

6. A method for identifying human hair follicle dermal stem cells, characterized in that: Detect whether the marker according to claim 1 or 2 is expressed in the cells to be identified.

7. A method for screening human hair follicle dermal stem cells, characterized in that: The method comprises screening a cell population for cells expressing the marker according to claim 1 or 2.

8. The method according to claim 7, characterized in that: The cell population is derived from hair follicles.

9. Use of the detection kit according to any one of claims 3 to 5 in the preparation of products for identifying, screening and preparing human hair follicle dermal stem cells.

10. A method for isolating human hair follicle dermal stem cells, characterized in that: The following steps are involved: Separate the dermis of the hair bulb of the hair follicle; Digesting the dermis with type I collagenase to obtain a digestion product; The digestion product is subjected to a first centrifugation to obtain a supernatant containing the human hair follicle dermal stem cells, and the centrifugal force of the first centrifugation is 5 g to 30 g.

11. The method according to claim 10, characterized in that: The method further comprises performing a second centrifugation on the supernatant to collect the precipitate, which is the human hair follicle dermal stem cells, wherein the centrifugal force of the second centrifugation is 200-400 g.

12. The method according to claim 10, characterized in that: The working concentration of the type I collagenase is 0.8-1.5 mg / mL.

13. A method for producing human hair follicle dermal stem cells, characterized in that: The method comprises obtaining and culturing human hair follicle dermal stem cells according to any one of the methods of claims 7 to 8 and claims 10 to 12.

14. A method for preparing cell secretions, characterized in that: The method comprises obtaining and culturing human hair follicle dermal stem cells according to any one of the methods of claims 7 to 8 and claims 10 to 12, and isolating the cell secretions.

15. Use of human hair follicle dermal stem cells obtained by any one of the methods of claims 7 to 8 and claims 10 to 13, or cell secretions obtained by the preparation method of claim 14 in preparing products for reconstructing skin or hair follicles, promoting hair growth, and treating skin diseases.

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